1. Field of the Invention
The field of the invention relates in general to at least one method and apparatus for the production of soluble MHC antigens and more particularly, but not by way of limitation, to at least one method and apparatus for the production of soluble Class I and II HLA molecules (i.e. sHLA). The field of the invention also includes such produced soluble Class I and II HLA molecules and their uses. According to the methodology of the present invention, the soluble Class I and II HLA molecules can be produced from either gDNA or cDNA starting material. One such exemplary, but non-limiting, use is the formation of a tetrameric sHLA (or other multimeric complex) complex which may be used to test immunogenicity of peptide ligands of interest—i.e. will a peptide ligand of interest provoke a CTL response and/or preferentially bind a CTL.
2. Brief Description of the Background Art
Class I HLA molecules are polymorphic human glycoproteins that endogenously bind and then extracellularly present peptide ligands to CD8+ T lymphocytes. Polymorphisms within the class I peptide binding groove are positioned to moderate ligand binding and presentation to such immune system cells. To date, the small quantities of natural ligands available to those of skill in the art has limited the understanding of precisely how polymorphism alters peptide binding and in turn, vaccine development and, more basically, if a peptide ligand of interest will provoke a CTL mediated immune response. In order to address the impact of polymorphism upon antigen presentation, the inventors developed the novel approach disclosed herein—i.e. that ligand presentation overlaps exist across the polymorphisms and that these overlaps distinguish divergent class I peptide binding grooves. Utilizing this novel approach and coupling it with a unique hollow-fiber cell culture scheme and utilizing a mass spectrometric ligand mapping approach, large quantities of peptides eluted from soluble class I and class II molecules can be obtained for detailed analyses, vaccine development, and functional testing.
Initially, peptide ligands were extracted from five different HLA-B15 allotypes and subsequently examined. Mapping and characterizing the ligands obtained from these allotypes demonstrated that they: (i) vary in length from 7 to 12 residues; (ii) are more conserved at their C termini than at their N-proximal residues; and (iii) are presented as overlaps contingent on C-terminal preferences. These results provide insight into class I and class II ligand loading not available via other methods, demonstrating that an elemental role is played by a peptide ligand's C terminus during endogenous binding and provides the starting material for a multimeric complex to be used to test the functionality of a peptide ligand of interest. The data obtained, and disclosed herein, validates, and illustrates the unique methods disclosed herein for the production of sHLA from either gDNA or DNA starting material and the uses to which this sHLA material may be put.
Class I and class II MHC molecules, designated HLA class I and class II, respectively, in humans, bind and display peptide antigens upon the cell surface. The peptides they present are derived from either normal endogenous proteins (“self”) or foreign proteins (“nonself”). Nonself proteins include items such as the products of malignant transformation or intracellular pathogens. In this manner, class I and class II molecules convey information regarding the internal fitness of a cell to CD8+ CTLs which are activated upon interaction with “nonself” peptides. Such activation may lead the CD8+ CTLs to kill and/or suppress a cell which is malignant or contains intracellular pathogens.
Examination of HLA by serologic and molecular methods by those of ordinary skill in the art continues to demonstrate that the class I HLA-A, B, and C molecules are encoded by the most polymorphic genes in mammals. Translating class I polymorphism into the tertiary structure of the class I molecule indicates that residues positioned to affect class I peptide presentation to T lymphocytes are most frequently affected by the mutagenic events which diversify class I loci. Throughout the world, HLA class I molecules exhibit a high degree of polymorphism that is generated by systematic recombinatorial events and collectively allows for the presentation of a vast array of different peptides. Depending upon allelic composition, two individuals' molecules may not necessarily bind the same peptides with equal affinity or even at all.
While the general structure and function of MHC class I molecules has been reasonably well studied and established, their polymorphic nature and how they specifically influence the capacity of class I in peptide binding and presentation remains an issue of persistent inquiry by those of ordinary skill in the art. The nature of precise overlaps in peptide binding specificity to HLA class I is particularly ill-defined at the current time due to the complexity of peptides bound. For example, this and other issues must be clarified in order to effectively pursue vaccines capable of eliciting protective CTL responses across an extensive population range. Unraveling the functional significance of class I polymorphism is an important issue that requires an understanding of how the mutagenic events diversifying the class I binding groove differentially moderate the presentation of peptide ligands.
The heavy chains of class I molecules are encoded within the MHC and, upon assembling into heterodimers with the light chain, β2m, are responsible for selectively gathering endogenously processed peptides. Once peptides are collected, mature class I molecules transport the bound peptides to the cell surface where receptors on CD8+ T lymphocytes engage the class I molecules to inspect the ligands. CTLs may then be triggered by class I molecules bearing virus or tumor-derived peptides.
With respect to the background art, hereafter numerous references are disclosed which detail one or more aspects of the background art as it relates to the novel methods and uses of the present invention. As such, each reference listed should be understood as being wholly incorporated by reference herein in their entirety as though the reference were fully transcribed herein. In this manner, one of skill in the art given the present specification would be fully informed and could truly appreciate the novel and unprecedented nature of the invention(s) disclosed and taught herein. The full and complete citations for each reference are appended hereto after the detailed description and before the claims.
The class I molecules expressed upon the nucleated cells of all vertebrate systems studied to date (for example, Ennis et al. 1988; Winkler et al. 1989; Kaufman et al. 1990; Lawlor et al. 1990; Trowsdale 1995; Prilliman et al. 1996; Antao et al. 1999) are heterodimers composed of a glycosylated 45 kDa heavy chain (α-chain) and a 12 kDa light chain (β2m). In humans, heavy chains are encoded at 3 loci (B, C, and A) within the MHC on the short arm of chromosome 6 (FIG. 1A). FIG. 1B illustrates each α-chain comprised of α1, α2, and α3 domains, as well as a transmembrane domain, which tethers the molecule to the cell surface and a short C-terminal cytoplasmic domain (Björkman and class I location and heavy chain coding region).
X-ray crystallography (Björkman et al. 1987a; Madden et al. 1991; Saper et al. 1991; Madden et al. 1992; Collins et al. 1995; Reid et al. 1996; Smith et al. 1996a; Smith et al. 1996b; Glithero et al. 1999) has illustrated details of the structural relationship of the extracellular α-chain domains and β2m (FIG. 2). The membrane-proximal domains, α3 and β2m, associate in an immunoglobulin fold structure. The membrane-distal α1 and α2 domains together create a closed basket-like structure that sits atop the α3 and β2m structure (FIG. 2A). It consists of two α-helical “walls” with a “floor” created by eight anti-parallel β-sheets (Björkman et al. 1987a; Madden 1995; and FIGS. 2, B and C). In the earliest studies, detection of electron density situated in the α1/α2 groove helped to clarify the experimentally-suspected occupancy of peptide fragments 8-10 residues long and thus the function of class I molecules in presenting such peptides upon the cell surface (Björkman et al. 1987b). Initial crystallographic studies designated six subsites, A-F (FIG. 3), or “specificity determining pockets,” that constitute the peptide binding groove (Garrett et al. 1989; Saper et al. 1991; Fremont et al. 1992; Matsumura et al. 1992; Elliott et al. 1993; Young et al. 1995). In addition to crystal structure analyses, thermodynamic stability studies indicate that networks of hydrogen bonds to structural residues lining the A- and F-pockets, which lie at opposite ends of the groove, serve to fasten a peptide by its N and C termini, respectively (Bouvier and Wiley 1994); these two pockets are thus implicated in the fixed orientation of a peptide within the binding groove.
Class I molecules primarily associate with peptide fragments, thus forming α-chain/β2 m/peptide trimolecular complexes, via an endogenous processing pathway during their assembly (Germain 1994; Heemels and Ploegh 1995; Lehner and Cresswell 1996; York and Rock 1996; Pamer and Cresswell 1998); in fact, the very binding of peptides is essential for the stabilization and expression of these molecules (Ljunggren et al. 1990; Townsend et al. 1990; Elliott 1991). The class I α-chain and β2m are cotranslationally translocated into the ER lumen (Townsend et al. 1990; Germain and Margulies 1993; Neefjes et al. 1993), where the α-chain remains anchored to the ER membrane and is stabilized prior to association with β2m and/or peptide through interactions with various chaperone proteins, including BiP, ER-60, calnexin, calreticulin, and tapasin (NöBner and Parham 1995; Sadasivan et al. 1996; Suh et al. 1996; Spee and Neefjes 1997; Harris et al. 1998; Lindquist et al. 1998). Although several alternative proteolytic processing and transport pathways, certainly exist (for example, Hsu et al. 1991; Henderson et al. 1992; Kozlowski et al. 1992; Roelse et al. 1994; Snyder et al. 1994; Ferris et al. 1996; Craiu et al. 1997; Luckey et al. 1998; Mosse et al. 1998; Wang et al. 1998; Young et al. 1998), it is believed that the majority of peptides the nascent class I molecules interact with are delivered to the ER in a distinct series of events.
Proteins in the cytoplasmic compartment are first enzymatically degraded into peptides of relatively uniform length by an ATP-dependent proteasome complex (Coux et al. 1996). Some proteasome components include the IFN-γ inducible subunits LMP2 and LMP7; these are themselves encoded within the MHC (Gaczynska et al. 1994). The typically nonameric fragments produced are then actively conveyed across the ER membrane via a dimer of TAP1/TAP2, an MHC-encoded ATP-binding cassette transporter (Monaco et al. 1990; Parham 1990; Grandea III et al. 1995). Once inside the ER, a processed peptide can be captured within the α1/α2 groove of a class I molecule and a stable trimer is formed (Germain and Margulies 1993; Smith et al. 1995). This trimeric α-chain/β2m/peptide complex is then transported through the Golgi complex and ultimately to the extracellular surface. The processes of class I assembly and transport are summarized in FIG. 4.
Following cell surface localization, mature complexes of class I bearing peptide antigens become available for interaction with receptors on monocytes, B and T lymphocytes, and NK cells (Townsend and Bodmer 1989; Yokoyama 1993; Lanier and Phillips 1996; Borges et al. 1997; Cosman et al. 1997). Their primary, and to date most thoroughly examined, natural receptor appears to be the TCR of T lymphocytes bearing the CD8 heterodimer. Site-directed mutagenesis and crystallographic studies indicate that the Vα and Vβ domains of heterodimeric TCRs associate in a diagonal fashion across the top surface of the structure formed by MHC α1 and α2 (Hogan et al. 1988; Lombardi et al. 1991; Moots 1993; Tussey et al. 1994; Garboczi et al. 1996; Garcia et al. 1996; Parham 1996; Björkman 1997; Smith and Lautz 1997; Manning et al. 1998). The hypervariable CDRs of Vα/Vβ contact specific regions of this interface (FIG. 5). Both precursor and effector CTLs are defined as being class I-restricted in that they are only capable of recognizing and responding to antigens displayed in the context of these molecules (Zinkemagel and Doherty 1974).
Since the antigens presented to CD8+ T lymphocytes are predominantly obtained through the processing of intracellular proteins as previously described, class I molecules figuratively serve as external banners that advertise the inner contents of the cells. Indeed, these antigens of peptide ligands indicate to the immune system as a whole which cells are to be eliminated and/or protected. Malignancies and/or pathogens effectively use this system to camouflage their existence and thereby escape detection and elimination by CD8+ CTLs, for example.
Thymic education of lymphocytes prevents activation in response to characteristic cell-derived peptides (Robey and Fowlkes 1994), but peptides acquired through the degradation of atypical proteins are recognized and induce cytolysis (Townsend et al. 1985; Gotch et al. 1988; Walker et al. 1988; Clark et al. 1995). Therefore, it is not surprising that CD8+ T lymphocytes play a critical role in controlling and/or eliminating infected and neoplastic cells.
CD8+T lymphocytes are implicated in immunity to pathogens such as viruses, which are intracellular invaders that utilize the host cell's biosynthetic machinery to produce their own foreign proteins (Yap et al. 1978; Lin and Askonas 1981; Jamieson et al. 1987; Harty and Bevan 1992; Riddell et al. 1992; Kulkarni et al. 1995; Heslop et al. 1996; Schmitz et al. 1999). CTL responses are likewise extended to include stimulation by aberrant proteins such as those associated with malignancy (Vose and Bonnard 1982; Muul et al. 1987; Coulie et al. 1992; Melief 1992; Kittlesen et al. 1998; Shichijo et al. 1998). In fact, class I molecules are capable of binding and presenting to CTLs any protein introduced into the endogenous processing pathway by either natural or artificial means (Gooding and O'Connell 1983; Moore et al. 1988; Yewdell and Bennink 1990; Bertoletti et al. 1991; Donnelly et al. 1993; Ikonomidis et al. 1994; Ballard et al. 1996; Day et al. 1997; Goletz et al. 1997; Kim et al. 1997). This knowledge serves as a motivating factor behind the development of both protein/peptide-based vaccines and other therapeutics intended to elicit protective CTL responses to microbial pathogens and other abnormalities, which otherwise remain cytoplasmically concealed from detection.
Fully understanding the role of class I molecules in ligand presentation as described above is complicated by α-chain polymorphism. Class I structural differences resulting from genetic variation confer extreme heterogeneity upon regions of the molecule that interact with peptides. The knowledge of how polymorphism specifically impacts the natural presentation of peptide epitopes upon the cell surface is consequently limited.
Class I MHC polymorphism was first documented in mice (Gorer 1936; Gorer 1937; Nathenson et al. 1981) and next studied in humans by serology (Dausset 1958; Payne and Hackel 1961; van Rood 1969); however, first protein and then DNA sequencing studies precisely demonstrated this genetic variability to be most concentrated throughout the exons of the α1 and α2 heavy chain domains at positions affecting amino acid residues that line the walls and floor of the previously described peptide binding groove (Orr et al. 1979; Tragardh et al. 1979; Rojos et al. 1987; Parham et al. 1988; Parham et al. 1989; Parham et al. 1995). It is the more centrally-located binding pockets (B-E), together with specific residues within the F-pocket, that appear to be the most polymorphic (Chelvanayagam 1996; Kostyu et al. 1997). Changes in the physicochemical properties of amino acid side chains within the groove can influence the stability with which given peptides interact during the assembly of α-chain/β2m/peptide trimers (Matsui et al. 1993; Rohren et al. 1993; Salter 1994; Young et al. 1995). Therefore, despite the overall structural conservation illustrated among class I α-chains (Björkman and Parham 1990; Madden 1995), their peptide binding grooves can vary drastically from one allelic form to another; as a result various isoforms are capable of associating with distinct arrays of peptides (Elliott et al. 1993; Smith et al. 1995; Smith et al. 1996b).
The characteristic polymorphism observed among class I molecules is thought to originate primarily through recombination and gene conversion (Kuhner et al. 1991; Parham et al. 1995); point mutations are believed to contribute more rarely to the pool of new alleles continually arising (Parham et al. 1989). Individuals inherit a set of three class I genes from each parent, and since their expression is codominant, a single person may therefore display up to six different HLA class I molecules upon his or her nucleated cells. From these alleles, new forms evolving progressively within populations can be passed on to subsequent generations and likewise serve as templates upon which yet further diversity may be introduced. This occurs through events such as single or double recombination (Parham et al. 1988) or nonreciprocal exchanges between cis-oriented gene segments during gene conversion (Parham 1992). Serological cross-reactivity studies, as well as locus-specific PCR amplification and sequencing analyses, have verified the existence of allelic subtypes, or allotypes, of closely related alleles that appear to have arisen from a common ancestral template by these molecular mechanisms (for example, Payne et al. 1978; Ooba et al. 1989; Hildebrand et al. 1994; Prilliman et al. 1996). While both inter- and intra-locus genetic events may give rise to polymorphism, the latter is most commonly observed; alleles at a locus generally tend to be more closely related to one another than to those present at other loci (Parham et al. 1988; Parham et al. 1995). The forces driving HLA class I polymorphism are believed to be those of overdominant or balancing selection (Hughes and Nei 1988; Hughes and Yeager 1998); this is based upon values of dN>dS within the coding regions of α1 and α2 for specificity determining pocket residues positioned to interact with the peptide binding groove, while the contrary (dS>dN) is observed among the remaining α1/α2 residues.
Considering the manner by which class I structural polymorphisms are generated and maintained demonstrates that HLA genetic variability affords both advantages and disadvantages. It is beneficial in ensuring that at least a small portion of the human population will possess class I molecules capable of: (i) binding immunogenic peptides derived from any given pathogen; (ii) presenting those peptides to CTLs; and (iii) evoking a protective imune response. In short, annihilation of the species is guarded against by molecular diversity (Parham 1992). The concept of heterozygote advantage through polymorphism as a mechanism for effectively allowing broader peptide binding abilities and thus broader CTL recognition of pathogenic peptides has been strongly emphasized from a statistical perspective (Hughes and Nei 1988; Nei and Hughes 1992). For example, HLA heterozygosity has been correlated with diminished progression to disease following HIV infection (Carrington et al. 1999). In addition, at the level of individual allotypes the “nonrandomness” with which certain polymorphisms are maintained within populations following their evolution supports positive natural selection. This might occur in response to specific pathogenic pressures, as seen in the strong association of the West African allele HLA-B*5301 with resistance to malaria (Hill et al. 1991; Hill et al. 1992), a parasitic illness endemic to West Africa. As mentioned previously however, the polymorphic nature of class I molecules results in divergent allotypes binding and presenting different peptides. CTLs thus focus on distinct portions of any given pathogen from one individual to another. Therefore, dissecting disease susceptibilities and resistances requires a grasp of how binding groove-localized amino acid variations specifically alter ligand presentation.
Numerous previous research endeavors have been directed toward understanding the structural and functional nature of peptides bound by HLA complexes; though some progress has been made in analyzing the manner that peptide binding is specifically influenced through α1/α2 substitutions, this knowledge remains limited and sometimes inconsistent. The full extent that polymorphisms dictate the degrees of ligand binding ability, stringency, and/or degeneracy (and subsequently cell surface presentation) has, as a result, not been adequately resolved. Similarly, the occurrence of overlapping ligands, or identical peptides presented across the binding groove polymorphisms of multiple distinct allotypes, remains to be explored.
Ideally, characterizing functional overlaps would provide an advantage not only to explore the general effects of binding groove architecture but more specifically to understand the similarities and/or differences of what is presented to CTLs by the class I molecules of genetically diverse individuals. In the search for answers to ligand binding influences by α-chain polymorphisms, methods including pooled Edman sequencing, mass spectrometric analysis, and binding/reconstitution assays have been employed. However, each approach bears its own strengths and limitations and none so far has been significantly successful in comparatively evaluating levels of functional overlap across class I polymorphisms. The importance of understanding peptide associations with polymorphic class I molecules at a level of complexity not necessarily afforded by the currently-defined strategies is thus underscored: epitope predictions based upon methods that fail to accurately assign possibilities for natural binding groove occupancy by either aberrant or low-abundance peptides of varying binding affinities interfered with detecting presentation overlaps among various HLA class I allotypes.
Early investigations of class I peptide ligands focused on simplifying the effects of polymorphism through establishing “motifs” (Rötzschke and Falk 1991; Rammensee et al. 1993; Engelhard 1994). Motifs have typically been established by purifying surface-expressed class I molecules from detergent lysates of either transformed cells or transfectants and extracting the bound ligands with either TFA or acetic acid. The resulting peptide pools are then subjected to consecutive cycles of N-terminal Edman degradation (Falk et al. 1991; Jardetzky et al. 1991).
The resultant motifs from N-Terminal Edman degradation are invariably nine amino acids in length and describe, as based upon relative yield increases per cycle, conserved “anchor” residues, or sites of stereochemical preferences, for peptides that are bound by a class I molecule. These anchors, typically appearing to involve both P2 and P9 of the ligands, are considered to be allele-specific and thus common among nearly all of the peptides bound by a given class I allotype. The remaining positions demonstrate no overriding amino acid preferences, although the motifs of a few molecules demonstrate anchors at P3 or P5 (Rammensee et al. 1997). The P2 anchor is assumed to associate with the B-pocket of the binding groove, while P9, the C-terminal residue assignment, associates with the F-pocket. “Auxiliary” or “secondary” anchors (alternative positions frequently defined by the occupancy of chemically similar residues) are additionally included in the motifs of some class I molecules (Rammensee et al. 1997). In general, a common interpretation of this type of data is that endogenous peptide binding and/or loading requires a nonamer with particular P2 and P9 anchors. As will be discussed, many searches (and consequent failed attempts) for putative viral or tumor class I-presented epitopes have subsequently been predicated upon nonameric templates with P2 and P9 anchor assignments.
Broad efforts have been focused upon establishing and analyzing motifs from natural class I ligands (for example, Huczko et al. 1993; Fleischhauer et al. 1994; Kubo et al. 1994; Barber et al. 1995; Steinle et al. 1995; Barber et al. 1996; Tzeng et al. 1996; Tieng et al. 1997; Yagüe et al. 1998). However, motifs fail to reflect the true complexity of peptides presented by divergent class I molecules. Drawbacks are evident in that numerous characterized peptide sequences that bind class I are greater than 9 residues long, with 14 being the largest identified to date (Engelhard 1994); The binding of longer ligands likely results from stable associations at anchor positions coupled with central protrusion of the peptide outward from the groove (Fremont et al. 1992; Guo et al. 1992; Madden et al. 1993). Furthermore, other examinations of specific peptides naturally presented by class I MHC of both humans and mice have indicated that some fail to comply with their respectively defined motif anchors (Calin-Laurens et al. 1993; Henderson et al. 1993; Sadovnikova et al. 1993; Kawakami et al. 1994; Urban et al. 1994; Malarkannan et al. 1996; Mata et al. 1998), thus suggesting that both length variance and “nonanchor” residues in the peptide could play significantly more prominent roles in binding than strictly accounted for by a given pooled motif alone. Studies have also indicated that low copy-number peptides, presented by only a small proportion of the total class I molecules expressed upon the cell surface, can successfully elicit CTL responses (Cox et al. 1994; Malarkannan et al. 1996; Wang et al. 1997). Issues such as these clearly reflect the limitations posed in applying Edman sequencing to the complex mixtures of peptides extracted from class I molecules (Stevanovic and Jung 1993). As a result, pooled Edman sequencing is therefore unable either to precisely characterize individual ligands or to effectively identify overlaps in ligand presentation.
Shortly after the debut of examining class I ligands by pooled Edman sequencing, the first reports of ligand analyses via mass spectrometric sequencing of organic ions from similarly-prepared class I ligand extracts began to emerge (Henderson et al. 1992; Hunt et al. 1992; Huczko et al. 1993; Appella et al. 1995). The utilization of MS/MS on a triple quadrupole mass spectrometer provided for the precise characterization of individual constituents at sub-picomolar levels from pools of class I peptides, in contrast to the picomolar detection limits of Edman analysis. Furthermore, LC/MS prior to this step allowed for complexity estimates to be made. For example, based upon the quantitation of 200 different peptides present within HLA-A*0201 extracts, extrapolation with regard to the peptides detected versus their respective contributions to the mass spectrometric TIC obtained indicated that at least 1,000 and perhaps as many as 10,000 unique peptides are bound by this class I allotype (Hunt et al. 1992). The ability to characterize ligands as such additionally assisted in being able to identify and sequence single specific epitopes from RP-HPLC fractions demonstrating biological activity via CTL assays (Rötzschke et al. 1990; Henderson et al. 1993; Kawakami et al. 1994; Skipper et al. 1996; Simmons et al. 1997; Wang et al. 1997; Hogan et al. 1998; Paradela et al. 1998). Examination of ligands by mass spectrometry was therefore an effective development in both starting to fill the gaps often present in pooled motifs and expediting the classification of ligands potentially bearing immunological significance.
However, the routine application of mass spectrometric techniques to class I ligand examination has remained relatively isolated; it is practiced in only a handful of laboratories (for example, Woods et al. 1995; Simmons et al. 1997; Flad et al. 1998; van der Heeft et al. 1998; Yagüe et al. 1998). This appears largely due to the inherent difficulties imposed in handling the small quantities of peptides extracted for study (Henderson et al. 1993; Appella et al. 1995; van der Heeft et al. 1998) as well as the notably tedious nature of the subsequent data processing (Papayannopoulos 1995; van der Heeft et al. 1998). Other issues concern the specific instrumentation and its mode of operation, since: (i) the ability to consistently identify peptides from particular extracts relies upon dependable RP-HPLC gradients for separation; and (ii) the possible ionization and/or detection interfaces function in distinct manners that can influence the data ultimately acquired (Chapman 1996; Watson 1997). In summary, understanding the impact of polymorphism upon the binding of endogenous peptdes has historically been limited by small amounts of ligands available for analyses.
What has not been accomplished yet by mass spectrometry is the systematic definition of peptides across diverse class I allotypes. Various in vitro assays have been developed to complement mass spectrometric approaches. The assays are performed using a number of different protocols with the common theme of assessing the relative abilities in vitro of synthetically defined peptides to associate with specific class I α-chains and β2m. In the case of binding assays, synthetic peptides as well as a peptide standard are incubated with purified class I complexes and tested in their capacities to competitively displace the ligands already bound (Chen and Parham 1989; Ruppert et al. 1993; Sette et al. 1994). This has likewise been performed by stripping ligands from class I complexes expressed on the cell surface by acid treatment and then incubating the cells with the synthetic peptides (Drijfhout et al. 1995). Another common approach to competitive binding assays involves FACS analysis with class I-specific MAbs after incubation of synthetic peptides with various cell lines including RMA-S (Townsend et al. 1989), T2 (Salter et al. 1985), Hmy2.C1R (Storkus et al. 1989), or 721.221 (Kavathas et al. 1980) transfected to express HLA α-chains (Huczko et al. 1993; Takamiya et al. 1994; Zeh III et al. 1994; Boisgerault et al. 1996; Konya et al. 1997). Alternatively, de novo reconstitution assays can be performed by incubating synthetic peptides with free α-chains and β2m and comparing the resultant quantities of free versus complexed α-chains (Tanigaki 1992; Fruci et al. 1993). Other methods of reconstitution have also been applied (Silver et al. 1991; Parker et al. 1994; Gnjatic et al. 1995; Robbins et al. 1995; Tan et al. 1997).
While these in vitro assays with synthetic ligands have led to the development of extensive “supermotifs”, or enhanced motifs that ascribe binding preferences to particular class I allotypes or groups of related allotypes (del Guercio et al. 1995; Sidney et al. 1996a; Sidney et al. 1996b), they present obvious limitations. A technical concern is that binding/reconstitution determinations are often based upon either “standard” probe peptides for each allotype examined or mean experimental values that can significantly differ from one laboratory or experiment to another. A specific illustration is based on the results generated by three different groups testing HLA-A*0201 with panels of synthetic peptides; the assignment of residues preferred or deleterious for binding at given positions varied widely (Ruppert et al. 1993; Parker et al. 1994; Drijfhout et al. 1995). In fact, only three residue similarities carried across all three studies.
Ruppert and colleagues and Drijfhout and colleagues each employed competitive binding assays; however, the former involved assessing the ability of synthetic peptides to inhibit binding of a radioiodinated standard peptide to membrane-extracted class I complexes, while the latter involved stripping peptides from class I complexes on B-LCLs and determining by FACS analysis the ability of synthetic peptides to inhibit binding of a different fluorescence-labelled standard. On the other hand, Parker and colleagues employed a reconstitution assay which measured the incorporation of radioiodinated β2m into complexes refolded using synthetic peptides and α-chains prepared from Escherichia coli inclusion bodies. Of perhaps greater significance, these types of assays fail to account for the processing/loading physiology of trimolecular complex formation within the cell (Hogan et al. 1988); indeed, differences have been documented in performing comparative examinations by pooled Edman sequencing and mass spectrometry upon naturally-extracted versus artificially bound peptides (Davenport et al. 1997). In one specific case, an immunodominant HIV peptide divulged through other mechanisms completely failed to demonstrate binding to its restricting allotype via an in vitro assay (Tsomides et al. 1991). Conclusions regarding ligand presentation that are drawn from binding/reconstitution assays thus cannot be applied generally without caveat.
Thus, the present invention(s) aim to provide a methodology for the production of soluble MHC Class I and II molecules from either gDNA or cDNA starting material such that the structural and functional impact of HLA class I polymorphism on peptide binding can be assessed and, in particular, to test how natural ligand presentation overlaps exist in varying degrees across the polymorphisms of divergent class I binding grooves. Furthermore, the soluble MHC molecules can be used in the functional testing of CTL binding assays and vaccine deveopment. Utilizing this specification, one of ordinary skill in the art will able to: (i) generate ligands and hence ligand maps from the peptide pools extracted from series of distinct yet related class I HLA-B15 allotypes; (ii) compare the different ligand maps to identify potentially shared elements; and (iii) characterize the elements identified to positively or negatively validate the occurrence of overlapping ligands. One of ordinary skill in the art, given the present specification, will also realize that the ability to produce soluble MHC molecules from either gDNA or cDNA starting material also will allow for other useful assay and vaccine development such as the functional testing of peptide ligands of interest to determine if, when, and how such peptide ligands provoke and/or stimulate an immune response. All of which is directed toward the goal of identifying candidate peptide ligands that may be used singly or together as a vaccine and/or immune system primer.